Bottom Line:
Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Affiliation: Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACTThis work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Mentions:
A crucial question amongst reports concerning the adherence and/or uptake of CNTs by cultured cells [1,7,9-18,37,43,65-73,75,77,78,81-84,86,88,91-94,113,114] is whether CNTs are toxic. Previously, we observed that the growth rates of HeLa cells incubated for 4 d in ~100 μg/mL HiPco SWNTs dispersed in a peptide solution or in media supplemented with serum were statistically similar to controls [19]. The evaluation of CoMoCAT DM-SWNTs also involved monitoring growth rates over the course of 4 d. First, there were no discernable differences in the morphologies of HeLa cells incubated in DM-SWNTs for 60 h (Figures 5 and 10B) relative to controls (Figure 10A; cells incubated in DMEM/FBS). Next, the growth rates of HeLa cells continuously exposed to DM-SWNTs were quantitated by calculating population double times (PDTs). A PDT is a measure of cell numbers at the early log growth phase and is used for comparisons of normal cell growth. PDTs were obtained from the slopes of the lines of a plot of the natural log of cell numbers versus time [120]. Figure 11 shows such a plot over a time period of 4 d for cells cultured in DM-SWNTs and control cells (DMEM/FBS only). For both samples, the respective number of HeLa cells counted on days 1, 2, 3, and 4 were statistically similar at a 95% confidence level. The control HeLa cell PDT was 27 h and was statistically similar to the PDT of 29 h observed with HeLa cells cultured in DM-SWNTs. In summary, the data from this sensitive test argue that our preparations and concentrations of purified CoMoCAT DM-SWNT dispersions do not affect HeLa cell growth rates.

Mentions:
A crucial question amongst reports concerning the adherence and/or uptake of CNTs by cultured cells [1,7,9-18,37,43,65-73,75,77,78,81-84,86,88,91-94,113,114] is whether CNTs are toxic. Previously, we observed that the growth rates of HeLa cells incubated for 4 d in ~100 μg/mL HiPco SWNTs dispersed in a peptide solution or in media supplemented with serum were statistically similar to controls [19]. The evaluation of CoMoCAT DM-SWNTs also involved monitoring growth rates over the course of 4 d. First, there were no discernable differences in the morphologies of HeLa cells incubated in DM-SWNTs for 60 h (Figures 5 and 10B) relative to controls (Figure 10A; cells incubated in DMEM/FBS). Next, the growth rates of HeLa cells continuously exposed to DM-SWNTs were quantitated by calculating population double times (PDTs). A PDT is a measure of cell numbers at the early log growth phase and is used for comparisons of normal cell growth. PDTs were obtained from the slopes of the lines of a plot of the natural log of cell numbers versus time [120]. Figure 11 shows such a plot over a time period of 4 d for cells cultured in DM-SWNTs and control cells (DMEM/FBS only). For both samples, the respective number of HeLa cells counted on days 1, 2, 3, and 4 were statistically similar at a 95% confidence level. The control HeLa cell PDT was 27 h and was statistically similar to the PDT of 29 h observed with HeLa cells cultured in DM-SWNTs. In summary, the data from this sensitive test argue that our preparations and concentrations of purified CoMoCAT DM-SWNT dispersions do not affect HeLa cell growth rates.

Bottom Line:
Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles.The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d.We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.

Affiliation:
Department of Chemistry, The University of Texas at Dallas, Richardson, TX 75080, USA. pantano@utdallas.edu.

ABSTRACTThis work concerns exposing cultured human epithelial-like HeLa cells to single-walled carbon nanotubes (SWNTs) dispersed in cell culture media supplemented with serum. First, the as-received CoMoCAT SWNT-containing powder was characterized using scanning electron microscopy and thermal gravimetric analyses. Characterizations of the purified dispersions, termed DM-SWNTs, involved atomic force microscopy, inductively coupled plasma - mass spectrometry, and absorption and Raman spectroscopies. Confocal microRaman spectroscopy was used to demonstrate that DM-SWNTs were taken up by HeLa cells in a time- and temperature-dependent fashion. Transmission electron microscopy revealed SWNT-like material in intracellular vacuoles. The morphologies and growth rates of HeLa cells exposed to DM-SWNTs were statistically similar to control cells over the course of 4 d. Finally, flow cytometry was used to show that the fluorescence from MitoSOXtrade mark Red, a selective indicator of superoxide in mitochondria, was statistically similar in both control cells and cells incubated in DM-SWNTs. The combined results indicate that under our sample preparation protocols and assay conditions, CoMoCAT DM-SWNT dispersions are not inherently cytotoxic to HeLa cells. We conclude with recommendations for improving the accuracy and comparability of carbon nanotube (CNT) cytotoxicity reports.